Cyclic AMP does not mediate the action of synthetic hypertrehalosemic peptides from the corpus cardiacum of Periplaneta americana

Two synthetic peptides identical to those present in the corpus cardiacum of the American cockroach, Periplaneta americana, were tested for their effect on the production of cyclic AMP and the activation of glycogen phosphorylase in cockroach fat body. The peptides activate glycogen phosphorylase and promote trehalose production in incubated tissue when calcium is included in the incubation medium, but have no obvious effect on cyclic AMP levels. The lack of effect of the peptides on cyclic AMP production was confirmed in a fragmented membrane preparation. By contrast, an aqueous extract of corpus cardiacum activates glycogen phosphorylase, promotes trehalose production and elevates cyclic AMP levels in incubated tissue; the extract also enhances cyclic AMP production in the fragmented cell membrane preparation. Observations on the nature of cyclic AMP production in cockroach fat body indicate that the adenylate cyclase has a requirement for GTP and magnesium ions, is stimulated by fluoride and forskolin and, therefore, is similar to the adenylate cyclase complex of other eukaryotes.The results suggest that increases in intracellular calcium concentrations may mediate the expression of hypertrehalosemic effects by the synthetic peptides.     

Lack of correlation between hormonal effects on cyclic AMP and glycogenolysis in rat liver.

Portions of liver were obtained by biopsy from rats infused with various concentrations of glucagon or epinephrine and analyzed for cyclic AMP, glycogen, phosphorylase activity, and glycogen synthetase I activity. The response of tissue cyclic AMP to glucagon or epinephrine was far less sensitive than other metabolic parameters; at certain lower doses of glucagon or epinephrine, glycogen decomposed without a simultaneous increase in the hepatic level of cyclic AMP. It is probable that hormonal activation of adenylate cyclase results in an increase of cyclic AMP only in its small “active” pool without detectable changes in its much larger inactive or bound pool. Though the active cyclic AMP is expected to be released into the circulation or to be labeled with [³H]adenine in preference to the inactive nucleotide, neither the increase of cyclic AMP in the vena cava in vivo nor the incorporation of [³H]adenine into tissue cyclic AMP in liver slices in vitro exhibited more sensitivity to glucagon than the hepatic level of cyclic AMP as a whole. Thus, it remains to be settled whether cyclic AMP is compartmentalized in the cell or plays no essential role in the stimulation of hepatic glycogenolysis induced by small doses of hormones.     

Regulation of glycogen metabolism in astrocytoma and neuroblastoma cells in culture.

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in aprallel with the medium glucose. The critical glucose concentration is 2.5 mM for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations. When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E1 is effective with the neuroblastoma cells.     

The actions of cyclic AMP on biosynthetic processes are mediated indirectly by cyclic AMP-dependent protein kinase.

Adrenalin and glucagon inhibit glycogen, fatty acid and cholesterol synthesis by elevation of cyclic AMP, activation of cyclic AMP-dependent protein kinase and increased phosphorylation of the rate-limiting enzymes of these pathways. Here, we review recent evidence which indicates that inhibition of these biosynthetic pathways in muscle, adipose tissue and liver is much more indirect than has previously been supposed. In particular, cyclic AMP-dependent protein kinase does not appear to inhibit glycogen synthase, acetyl-CoA carboxylase and HMG-CoA reductase by phosphorylating them directly. It appears to achieve the same end result by inactivation of the protein phosphatases which dephosphorylate these regulatory enzymes in vivo, although this has only been established definitively in the case of glycogen synthesis.     

Regulation of bacterial glycogen synthesis. Stimulation of glycogen synthesis by endogenous and exogenous cyclic adenosine 3':5'-monophosphate in Escherichia coli and the requirement for a functional CRP gene

In Escherichia coli cya mutants, deficient in adenylate cyclase (EC 4.6.1.1), basal cellular rates of glycogen synthesis were lower and the relative increases produced by exogenous cyclic adenosine 3',5'-monophosphate during growth on glucose were greater than in their respective parent strains. These observations provide strong evidence that endogenous cyclic AMP is one of the key regulators of glycogen synthesis in growing E. coli. In crp mutants, deficient in cyclic AMP receptor protein (CRP), the basal cellular rates of glycogen synthesis were much lower than in their respective parent strains. Stimulation of glycogen synthesis by exogenous cyclic AMP was markedly attenuated in the three crp mutants. Thus, stimulation of glycogen synthesis by either endogenous or exogenous cyclic AMP appears to require CRP. Functional CRP appeared to be required for all three responses observed after cyclic AMP addition: an abrupt step-up in the cellular rate of glycogen synthesis, a continuing exponential increase in rate, and a stimulation of the rate during a subsequent nitrogen starvation. To account for these responses, we derived a mathematical model in which the cyclic AMP-CRP complex regulates the differential rate of synthesis of an enzyme metabolizing an effector of the rate-limiting enzyme of glycogen synthesis.     

Exercise-induced increases in myocardial adenosine 3′,5′-cyclic monophosphate and phosphodiesterase activity

Numerous cellular biochemical events caused by hormones are mediated throught cyclic AMP. Although many changes occur in the cell during exercise that could be attributed to this nucleotide, little evidence is available implicating it as an important regulator of exercise metabolism. In this investigation it was found that a 60 min bout of treadmill exercise caused a 2.4-fold increase in myocardial cyclic AMP immediately following the work. Rather than the imemediate nucleotide hydrolysis that was expected, it was found that the elevated cyclic AMP level remained for approx. 24 h before returning to control levels. Cardiac glycogen fell to 30% of control after work but supercompensated 60% above control within 1 h following exercise. Therefore, cardiac cyclic AMP was elevated at a time when glycogen was being synthesized. Study of the temporal relationship between the exercise-induced increase in cyclic AMP and cyclic nucleotide phosphodiesterase indicated that the work caused an increase in the hearts' capacity to hydrolyze cyclic AMP. Measurement of heart phosphodiesterase at substrate concentrations of 1.0 and 100 μM produced significant increased in enzyme activity immediately following exercise which remained elevated for 48 h and was back to control activity 96 h following work. These data present a potentially fascinating model for the study of the dissociation between cyclic AMP, glycogenesis and elevations in phosphodiesterase activity in the heart.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: EFFECT OF HYPOTHERMIA ON ADENOSINE 3'',5'' MONOPHOSPHATE, GLYCOGEN PHOSPHORYLASE, GLYCOGEN SYNTHASE AND METABOLITES FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

Abstract —The accumulation of adenosine-3',5'-cyclic monophosphate (cyclic AMP) has been investigated in murine brain following electroconvulsive shock and decapitation. Animals were made hypothermic (20°C) to minimize the freezing time of the brain and to delay metabolic events. Cyclic AMP concentrations were decreased in the cerebral cortex of hypothermic rats and mice. Furthermore, the changes in cyclic AMP elicited by electroconvulsive shock and decapitation were delayed. In hypothermic animals, the metabolic rate as determined by high energy phosphate use was decreased to 65% of control values. The interconversions of the active and inactive forms of glycogen phosphorylase and glycogen synthase were sufficiently retarded in hypothermic animals to correlate with changes in cyclic AMP concentrations. The conversion of phosphorylase b to a and synthase a to b occurred when cyclic AMP concentrations had increased from 2 to 5 μmol/kg, following either electroconvulsive shock or decapitation. The results indicate that cyclic AMP plays a role in regulation of glycogen metabolism in cerebral cortex.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

Activation of Glycogen Phosphorylase by Cyclic AMP in Tetrahymena pyriformis1

Glycogen phosphorylase in Tetrahymena pyriformis was activated by a Mg²⁺ ATP-dependent process and this activation was further increased by the addition of cyclic AMP. When the enzyme activity in subcellular fractions was measured, it was largely associated with the glycogen fraction but was no longer activated by ATP and cyclic AMP. Mixing the glycogen fraction and cytosol fraction together restored the effects of ATP and cyclic AMP on phosphorylase activity. These findings suggest that glycogen phosphorylase associated with Tetrahymena glycogen granules may be regulated by cytosolic factor(s) with cyclic AMP.     

Hormonal regulation of glycogen synthase: Insulin decreases protein kinase sensitivity to cyclic AMP

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1–2 μM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 μM. Insulin (0–100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.     

Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

Background

Signaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state.

Results

The glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase.

Conclusion

The highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different sensitivities of these two enzymes may exemplify the adaptive strategies employed by liver and muscle cells to meet specific cellular demands.

     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Cytophotometric analysis of glycogen,protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G₂/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N⁶,O²-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G₀/G₁ and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

COMPARISON OF LEVELS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON-NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA

Primary cultures containing ≥99% neurons, ≥99% non-neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12-day chick embryos. Levels of cyclic AMP in the non-neuronal cells (～14 pmol/mg protein) were approximately 3-fold higher than levels in the neurons (～4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non-neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non-neuronal cells. Thus, contact between the neurons and non-neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal-glial interactions, such as the specific neuronal stimulation of non-neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [³H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non-neuronal cells varied inversely with cell density. [³H]Thymidine incorporation was independent of cell density in both neuronal and non-neuronal cultures. Parallel density-dependent decreases in cyclic AMP concentration and [³H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non-neuronal cell proliferation and cyclic AMP levels in these cultures.     

Exercise-induced increases in myocardial adenosine 3',5'-cyclic monophosphate and phosphodiesterase activity

Numerous cellular biochemical events caused by hormones are mediated through cyclic AMP. Although many changes occur in the cell during exercise that could be attributed to this nucleotide, little evidence is available implicating it as an important regulator of exercise metabolism. In this investigation it was found that a 60 min bout of treadmill exercise caused a 2.4-fold increase in myocardial cyclic AMP immediately following the work. Rather than the immediate nucleotide hydrolysis that was expected, it was found that the elevated cyclic AMP level remained for approx. 24 h before returning to control levels. Cardiac glycogen fell to 30% of control after work but supercompensated 60% above control within 1 h following exercise. Therefore, cardiac cyclic AMP was elevated at a time when glycogen was being synthesized. Study of the temporal relationship between the exercise-induced increase in cyclic AMP and cyclic nucleotide phosphodiesterase indicated that the work caused an increase in the hearts' capacity to hydrolyze cyclic AMP. Measurement of heart phosphodiesterase at substrate concentrations of 1.0 and 100 microM produced significant increases in enzyme activity immediately following exercise which remained elevated for 48 h and was back to control activity 96 h following work. These data present a potentially fascinating model for the study of the dissociation between cyclic AMP, glycogenesis and elevations in phosphodiesterase activity in the heart.     

Short-term stimulation of net glycogen production by insulin in rat hepatocytes

Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.     

Cytophotometric analysis of glycogen, protein and DNA of a glycogen-storing rat hepatoma (N13) cell line.

This study examines the behavior of glycogen-storing rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2'-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Glycogen metabolism and cyclic AMP levels in isolated islets of lean and genetically obese mice.

The levels of glycogen and cyclic AMP, incorporation of glucose into glycogen and activities of glycogen synthetase and phosphorylase were determined in pancreatic islets isolated from genetically obese mice and their lean litter-mates. Islets from obese mice had elevated glycogen levels, increased phosphorylase activity and an increased amount of glycogen synthetase in the physiologically more effective I-form, indicating an increased turnover of glycogen. There was no significant difference in cyclic AMP levels between islets of lean and obese mice, but inhibition of phosphodiesterase or stimulation of adenyl cyclase increased cyclic AMP levels more in obese than in lean mouse islets, indicating a more rapid turnover of cyclic AMP in the former. It is suggested that cyclic AMP stimulated phosphorolytic breakdown of glycogen may be one of the mechanisms responsible for the increased insulin secretory response to glucose observed in islets from genetically obese mice.     

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine. 30 seconds later uteri were frozen $\text{in}$$\text{situ}$ and assayed for cyclic AMP and glycogen phosphorylase. The cyclic AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50% of that in non-estrogen-treated controls. Catecholamine-induced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls. At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls. Estrogen regulates uterine β-adrenergic sensitivity but the time courses of estrogen effects on the cyclic AMP and glycogen phosphorylase response changes are different. Catecholamine-induced uterine cyclic AMP formation is biphasic: suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours. Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24–48 hours of estrogen. It is concluded that estrogen has independent effects on the β-adrenergic-glycogen phosphorylase activation pathway at two different points; one prior to cyclic AMP formation and another after cyclic AMP formation.